Heretofore, as techniques for detecting a protein, immunological analysis techniques utilizing an antibody with respect to the protein have been utilized widely. For example, with an ELISA technique, an object protein is quantitatively analyzed with a high sensitivity. Also, with a Western blotting technique, excellent separation competence of electrophoresis and high specificity of an antigen-antibody reaction are combined with each other, and a specific protein is thereby detected from a protein mixture. Further, in cases where a labeled antibody is caused to act upon a tissue sample, a localized state of an object protein within a tissue or within a cell is observed.
However, with the immunochemistry techniques described above, it is necessary to perform complicated operations, such as an antibody binding operation and a subsequent washing operation.
As techniques for observing movement of a protein within an organism, techniques utilizing various fluorescent proteins, typically a green fluorescent protein (GFP), have been known. The fluorescent proteins may be formed within an organism by genetic engineering and are therefore useful for observation of the localized state of an endogenous protein within a tissue or within a cell and the movement of the endogenous protein.
However, with the aforesaid techniques utilizing the fluorescent proteins, the object protein must be formed within the viable cell in the form having previously been fused with the fluorescent protein, and it is not always possible to visualize an exogenous antigen within the cell.
Various studies have been conducted for solving the problems described above. For example, a fluorescence resonance energy transfer technique, or the like, is often utilized for analysis of spatial close positions of labeled biomolecules or molecule groups in a biological system. The fluorescence resonance energy transfer technique is useful as means for verification with respect to various biological reactions or interactions, which are objects of interest, such as a protein-protein interaction, an antigen-antibody reaction in an immune reaction, a receptor-ligand interaction, hybridism of nucleic acid, and binding of a protein to nucleic acid.
In patent literature 1, a technique is described, wherein an immunological analysis reagent containing an antigen and an antibody, which have been joined together by a linker and have respectively been labeled with fluorescent substances, is used, and wherein detection is made on a real time basis in accordance with a competitive reaction with an exogenous antigen. It is described that, with the immunological analysis reagent described above, in cases where the competing exogenous antigen is not present, the fluorescence resonance energy transfer (FRET) arises due to the formation of an antigen-antibody complex. It is also described that, in cases where the competing exogenous antigen is present, since the competing exogenous antigen is bound with the antibody and since the FRET does not arise, the presence or absence of the exogenous antigen is detected quickly and in a simple manner.
However, with the technique described in patent literature 1, wherein the antigen-antibody complex is not fixed by a support, the problems occur in that a washing operation is not capable of being performed, and that the immunological analysis reagent diffuses and flows out little by little in an unclosed space. Therefore, the immunological analysis reagent is capable of being utilized only one time, and it is not always possible to use the immunological analysis reagent in applications in which the immunological analysis reagent is to be utilized repeatedly. Also, since the detection with the competition technique is made, the sensitivity is apt to become bad and is not sufficient for the detection of a trace quantity of the antigen. Further, the competition technique, wherein an analysis is made by later adding a different reagent, is not suitable for the analysis, for example, within the organism. Furthermore, in cases where two kinds of reactants are joined together via a linker as in the technique described in patent literature 1, the problems occur in that, since the linker must be designed at the time of every joining operation such that the reactants may act efficiently, antigen flexibility is lost, and the production cost becomes high. For repetitive utilization, the linker may be removed, and the two kinds of the reactants may be separated from each other and merely fixed to the support. In such cases, the distance between a YFP acting as a first luminous substance and a CFP acting as a second luminous substance is not capable of being rendered close. If a fixation quantity of the YFP and the fixation quantity of the CFP are increased for rendering the YFP and the CFP close to each other, the problems will occur in that the large quantity of the YFP and the large quantity of the CFP become close to each other, and in that a signal-to-noise ratio becomes markedly low.
As a technique for fixing an antigen onto a solid layer by the utilization of the antigen-antibody reaction, a technique referred to as molecular imprinting is disclosed in, for example, patent literature 2. Specifically, it is described that a molecular imprint gel is obtained with processing, wherein a complex of a monomer or a polymer, into which a ligand with respect to an object molecule has been introduced, and the object molecular are subjected to a reaction with a polymeric crosslinking agent, where a gel containing a complex of the ligand and the object molecule is thereby prepared, and wherein the object molecule is then removed from the gel. Also, it is disclosed that, in cases where an AFP acting as an antigen is added to a gel carrier, which contains an anti-AFP antibody and lectin, the gel contracts due to the binding of the anti-AFP antibody and lectin with the AFP. It is described that the presence of the AFP (the object molecule) is detected in accordance with the swelling and the contraction of the gel.
However, since the antigen-antibody complex is fixed to the support by polymerization, in order for the antigen to arrive at the antibody, the antigen must enter into the crosslinked gel matrix structure. Therefore, a long time is required before the antigen and the antibody undergo the reaction. In order for the substance to be recognized reliably, the antigen in a markedly high concentration must be brought into contact with the antigen-antibody complex for a length of time ranging from two hours to four hours. Also, for the same reasons, at the time of washing and removal of the antigen, the antigen is not capable of being removed efficiently, and response characteristics are apt to become bad. Therefore, in cases where the fluorescence resonance energy transfer technique is applied to the molecular imprinting technique described above, since an alteration in distance between a fluorescent label site and a fluorescence recognition site for recognition of the fluorescence, which is produced by the fluorescent label site, is markedly slow, the problems occur in that the fluorescent label site and the fluorescence recognition site are always in a close state as for a short length of time (for example, a length of time of approximately five minutes), in that an alteration in fluorescence intensity is thus not capable of being analyzed, and in that the detection is not made quickly and with a high sensitivity.    Patent literature 1:
Japanese Unexamined Patent Publication No. 2007-40834    Patent literature 2:
Japanese Unexamined Patent Publication No. 2006-138656